白花丹参治疗TAO的有效成分筛选及其多途径作用机制研究

Thromboangiitis obliterans (TAO), also called Buerger's disease, is a type of vasculitis in which there is a recurring progressive inflammation and thrombosis (clotting) of small and medium arteries and veins of the hands and feet and can lead to serious complications if not treated promptly. As a folk herbal medicine, the root of Salvia miltiorrhiza Bunge var. alba C.Y.wu et H.W.Li has been used to treat TAO for a long time, and its clinical curative effect is better than that of the root of S. miltiorrhiza Bunge. But the reports of research on its effective constituents and mechanism of treatment on TAO have not been retrieved. We had done a bit more research on the root of S. miltiorrhiza Bunge var. alba. 31 compounds,together with 4 new compounds, were isolated and identificated from it. We have prepared the total phenanthraquinones and total phenolic acids, investigated their treatment effects on experimental TAO model rat and suggest that they can cause a significant therapeutic effect on experimental TAO model rats by inhibiting platelet aggregation and enhancing anti-thrombus function of vessel endothelia. We also had cultured rat aortic endothelial cells (RAEC) and tried establishing RAEC injuried model in vitro induced by LPS. Therefore, on the basis of the above research, in this project, firstly, we plan to isolate and obtain much more ammount of the 7 main components of the root of S. miltiorrhiza Bunge var. alba and investigate their pharmacodynamic action on experimental TAO rat. Secondly, according to the pathological character of TAO, By investigating their action of anti-inflammation, antiplatelet aggregative activity, thrombolysis and protection and repair of injuried RAEC, we plan to study on the bioactivity of the 35 compounds obtained from the root S. miltiorrhiza Bunge var. alba (including 4 new compounds), and research on the action mechanism of the 4 new compounds and 7 main components isolated from the root S. miltiorrhiza Bunge var. alba. At last, we hope to find the effective constituents of treatment on TAO and interpret the mechanism of action on TAO in many ways in order to provide the basis of S. miltiorrhiza Bunge var. alba for its clinical applation.

血栓闭塞性脉管炎(TAO)是一种以非化脓性炎症和动脉腔内血栓形成为特征的慢性闭塞性疾病。作为民间用药，白花丹参已长期用于治疗TAO，且疗效优于紫丹参，但其治疗TAO的有效成分及作用机制尚无报道。申请人已对白花丹参进行了较深入的研究，分离鉴定了35个化合物，其中4个新化合物；制备了白花丹参总菲醌和总酚酸，研究了这两个部位对TAO大鼠模型的治疗作用，疗效较好；探讨了大鼠动脉内皮细胞培养及其细胞损伤模型的建立。在此基础上，本项目拟：富集白花丹参的7种主要成分，进行TAO大鼠模型的体内药效学研究；结合TAO的病理特点，从抗炎、抗血小板、溶栓、对大鼠动脉内皮细胞损伤的保护和修复几个方面对35个化合物(其中4个新化合物)进行体外抗TAO的生物活性评价，并对其中的4个新化合物和富集的7种主要成分进行体外作用机制探讨，筛选白花丹参治疗TAO的有效成分，阐明其治疗TAO的多途径作用机理，为临床应用提供依据。

本项目对白花丹参化学成分进行了分离和鉴定，先后从中分离得到了45个化合物，包括24个丹参酮类成分，21个酚酸类成分，并对其中2个新化合物进行了结构研究报道。对白花丹参药材中丹酚酸B转化为丹酚酸A的工艺条件进行了研究，得到了稳定性及转化率均较高的丹酚酸A转化工艺，扩大了丹酚酸A的来源；对转化处理后白花丹参药材中丹酚酸A的提取和纯化工艺、白花丹参原药材中丹酚酸B的纯化工艺进行了研究，优化了其提取和纯化工艺条件；采用人主动脉内皮细胞、人THP-1巨噬细胞、EA.hy926人脐静脉细胞，对白花丹参中分离得到的部分丹参酮类和酚酸类成分从细胞因子及其蛋白表达的水平上进行了保护血管内皮细胞、抗炎、抗血栓形成的生物活性评价及其作用机制研究，结果这两类成分均具有比较好的抗炎活性，其中多个化合物的抗炎活性优于目前临床上使用的丹参酮IIA或丹酚酸B，如丹参醇A和紫草酸；均能显著降低EA.hy926细胞对PAI-1的表达、增加t-PA的表达，具有较好的抗血栓形成活性，其中有些化合物的抗血栓形成活性与阿司匹林的抗血栓活性相当，如去羟基新隐丹参酮和原紫草酸。此外，对白花丹参总提取物、总丹参酮、总酚酸、丹参酮IIA、丹酚酸A、丹酚酸B治疗TAO模型大鼠的药效学及作用机理进行了研究，结果表明白花丹参提取物及其主要成分对TAO大鼠均具有较好的治疗作用。以上研究的完成，比较系统地阐明了白花丹参治疗TAO的有效物质基础及作用机制，为临床应用白花丹参治疗TAO提供了科学依据，也为白花丹参的资源利用和开发奠定了基础。